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Eclipse ti2000

Manufactured by Nikon
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The Eclipse Ti2000 is a microscope system designed for advanced research applications. It features a modular design and supports a wide range of imaging techniques, including fluorescence, phase contrast, and differential interference contrast. The Eclipse Ti2000 is equipped with precise motorized controls and integrated image acquisition software, enabling researchers to capture high-quality, reproducible data.

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Lab products found in correlation

Hoechst 33342, by Thermo Fisher Scientific (3 mentions) Nis elements software, by Nikon (3 mentions) Plan apo vc 100, by Nikon (2 mentions) Fluosphere, by Thermo Fisher Scientific (2 mentions) Anti iba1, by Fujifilm (2 mentions) Hoechst 33258, by Merck Group (2 mentions) D glucose, by Merck Group (2 mentions) Nunc lab tek 2 chamber slide, by Thermo Fisher Scientific (1 mentions) Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (1 mentions) Fm4 64, by Thermo Fisher Scientific (1 mentions) Plan apo vc, by Nikon (1 mentions)

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Ti2000

6 protocols using eclipse ti2000

1

hMSCs Seeding and Imaging

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hMSCs were seeded on Nunc Lab‐Tek II chamber slides (Thermo Scientific) at 8,000 cells/cm2 and treated as described previously for flow cytometry. After 24 hr, cells were fixed in 4% paraformaldehyde and mounted with ProLong Gold Antifade Mountant with DAPI (4′,6‐diamidino‐2‐phenylindole, Invitrogen). Phase and fluorescence images were obtained using a Nikon Eclipse Ti2000 inverted microscope.
Malcolm D.W., Sorrells J.E., Van Twisk D., Thakar J, & Benoit D.S. (2016). Evaluating side effects of nanoparticle‐mediated siRNA delivery to mesenchymal stem cells using next generation sequencing and enrichment analysis. Bioengineering & Translational Medicine, 1(2), 193-206.
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2

Visualizing C. difficile Sporulation

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C. difficile strains were grown for 18 hours on 70:30 media, harvested into 1 ml of PBS containing 1 µg ml–1 FM4–64 (Molecular Probes) and 15 µg ml−1 Hoechst 33342 (Molecular Probes) and visualized on agarose pads as previously described (Fimlaid et al., 2013 (link)) except with the following modifications. DIC and fluorescence microscopy were performed using a Nikon PlanApo Vc 100 × oil immersion objective (1.4 NA) on a Nikon Eclipse Ti2000 epifluorescence microscope. Five fields for each sample were acquired with an EXi Blue Mono camera (QImaging) with 2×2 binning, hardware gain setting of 2.6, and driven by NIS-Elements software (Nikon). Images were subsequently imported into Adobe Photoshop CS6 for pseudocoloring and minimal adjustments in brightness/contrast levels. Phase-contrast microscopy for imaging the samples used for sporulation assays was performed as previously described (Putnam et al., 2013 (link)).
Pishdadian K., Fimlaid K.A, & Shen A. (2014). SpoIIID-mediated regulation of σK function during Clostridium difficile sporulation. Molecular microbiology, 95(2), 189-208.
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3

Live Cell Fluorescence Microscopy of Sporulating Bacteria

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Live cell fluorescence microscopy was performed using Hoechst 33342 (Molecular Probes; 15 µg/ml) and mCherry protein fusions to localize IVA. Samples were prepared on agarose pads as previously described (39 (link)) except that the samples were not imaged until 30 min after harvesting, since this time frame allowed for reconstitution of mCherry fluorescence signal in the anaerobically growing bacteria. Sporulating cells were exposed to ambient oxygen for a maximum of 80 min to minimize DNA fragmentation; no cell lysis was observed under these conditions. Differential interference contrast (DIC) and fluorescence microscopy were performed using a Nikon PlanApo Vc 100× oil immersion objective (1.4 numerical aperture [NA]) on a Nikon Eclipse Ti2000 epifluorescence microscope. An EXi Blue Mono camera (QImaging) with a hardware gain setting of 2.0 was used to acquire multiple fields for each sample in 14-bit format with 2-by-2 binning using NIS-Elements software (Nikon). The Texas Red channel was used to acquire images after a 300- to 400-ms exposure, 75- to 90-ms exposures were used to visualize the Hoechst stain, and ~10- to 20-ms exposures were used for DIC microscopy. Twenty-megahertz images were subsequently imported into Adobe Photoshop CS6 for minimal adjustments in brightness/contrast levels and pseudocoloring.
Ribis J.W., Ravichandran P., Putnam E.E., Pishdadian K, & Shen A. (2017). The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation. mSphere, 2(5), e00315-17.
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4

Live Cell Fluorescence Microscopy of C. difficile

Cited 2 times
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Live cell fluorescence microscopy was performed using Hoechst 33342 (Molecular Probes, 15 μg/mL) and 1 μg/mL FM4–64 membrane stain (Molecular Probes) as previously described (Fimlaid et al., 2013 (link), Fimlaid et al., 2015b (link)). Briefly, 23 hr sporulating cultures of C. difficile strains were harvested into 1 mL PBS, pelleted, and resuspended into 100 μL PBS containing the dyes listed above. Live bacterial suspensions (4 μL) were added to a freshly prepared 1% agarose pad.
DIC and fluorescence microscopy was performed using a Nikon PlanApo Vc 100x oil immersion objective (1.4 NA) on a Nikon Eclipse Ti2000 epifluorescence microscope. Multiple fields for each sample were acquired with an EXi Blue Mono camera (QImaging) with a hardware gain setting of 1.0 using the NIS-Elements software (Nikon). Images were subsequently imported into Adobe Photoshop CC 2015 for minimal adjustments in brightness/contrast levels and pseudocoloring. The percentage of sporulating cells of wildtype, ΔIID, ΔIIP, and ΔIIQ that had completed engulfment was determined from 6–10 fields from one to two biological replicates. A minimum of 200 cells were counted per strain.
Ribis J.W., Fimlaid K.A, & Shen A. (2018). Differential requirements for conserved peptidoglycan remodeling enzymes during Clostridioides difficile spore formation. Molecular microbiology, 110(3), 370-389.
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5

Quantifying Microglial Phagocytic Activity

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The phagocytic activity of adult microglia in the hippocampus of acute brain slices was analyzed as described61 (link). Brains from LysM-Cre+/GRNF/F or LysM-Cre−/GRNF/F mice were washed in carbogen-saturated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 126; KCl 2.5; MgSO4 1.3; CaCl2 2.5; NaH2PO4 1.25; NaHCO3 26; D-glucose 10; pH 7.4 (all from Sigma). Coronal slices (130 μm) were prepared using a vibratome (Microm, Walldorf, Germany) at 4°C, and allowed to rest in ACSF buffer at room temperature for 1 h before incubation with red fluorescent carboxylated microspheres (1 μm diameter, FluoSpheres®, Invitrogen, 1:100) in PBS (Cellgro) containing 4.5 g l−1 D-glucose (Sigma) for 60 min at 37°C. The slices were washed and fixed with 4% PFA. To visualize microglia, slices were permeabilized (2% Triton-X, 2% BSA, 10% donkey serum in PBS) and incubated with anti-Iba-1 (1:750, Wako), followed by donkey anti-rabbit Alexa 488 (1:250, Invitrogen) and Hoechst 33258 (Sigma-Aldrich; 1:10000). Imaging was performed with a Nikon ECLIPSE Ti 2000 spinning disk confocal microscope with a 20x objective. Slice-spanning Z-stack images (1 μm step-size) were acquired and analyzed using ImageJ MacBiophotonics cell counter plugin.
Minami S.S., Min S.W., Krabbe G., Wang C., Zhou Y., Asgarov R., Li Y., Martens L.H., Elia L.P., Ward M.E., Mucke L., Farese RV J.r, & Gan L. (2014). Progranulin Protects against Amyloid β Deposition and Toxicity in Alzheimer’s Disease Mouse Models. Nature medicine, 20(10), 1157-1164.
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6

Quantifying Microglial Phagocytic Activity

Cited 1 time
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The phagocytic activity of adult microglia in the hippocampus of acute brain slices was analyzed as described61 (link). Brains from LysM-Cre+/GRNF/F or LysM-Cre−/GRNF/F mice were washed in carbogen-saturated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF) containing (in mM): NaCl 126; KCl 2.5; MgSO4 1.3; CaCl2 2.5; NaH2PO4 1.25; NaHCO3 26; D-glucose 10; pH 7.4 (all from Sigma). Coronal slices (130 μm) were prepared using a vibratome (Microm, Walldorf, Germany) at 4°C, and allowed to rest in ACSF buffer at room temperature for 1 h before incubation with red fluorescent carboxylated microspheres (1 μm diameter, FluoSpheres®, Invitrogen, 1:100) in PBS (Cellgro) containing 4.5 g l−1 D-glucose (Sigma) for 60 min at 37°C. The slices were washed and fixed with 4% PFA. To visualize microglia, slices were permeabilized (2% Triton-X, 2% BSA, 10% donkey serum in PBS) and incubated with anti-Iba-1 (1:750, Wako), followed by donkey anti-rabbit Alexa 488 (1:250, Invitrogen) and Hoechst 33258 (Sigma-Aldrich; 1:10000). Imaging was performed with a Nikon ECLIPSE Ti 2000 spinning disk confocal microscope with a 20x objective. Slice-spanning Z-stack images (1 μm step-size) were acquired and analyzed using ImageJ MacBiophotonics cell counter plugin.
Minami S.S., Min S.W., Krabbe G., Wang C., Zhou Y., Asgarov R., Li Y., Martens L.H., Elia L.P., Ward M.E., Mucke L., Farese RV J.r, & Gan L. (2014). Progranulin Protects against Amyloid β Deposition and Toxicity in Alzheimer’s Disease Mouse Models. Nature medicine, 20(10), 1157-1164.
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